The mechanisms by which regulatory T cells (T regs ) suppress autoantibody production are unclear. Here we have addressed this question using transgenic mice expressing model antigens in the kidney. We report that T regs were essential and sufficient to suppress autoreactive B cells in an antigen-specific manner and to prevent them from producing autoantibodies. Most of this suppression was mediated through the inhibitory cell-surface-molecule programmed death-1 (PD-1). Suppression required PD-1 expression on autoreactive B cells and expression of the two PD-1 ligands on T regs . PD-1 ligation inhibited activation of autoreactive B cells, suppressed their proliferation, and induced their apoptosis. Intermediate PD-1 + cells, such as T helper cells, were dispensable for suppression. These findings demonstrate in vivo that T regs use PD-1 ligands to directly suppress autoreactive B cells, and they identify a previously undescribed peripheral B-cell tolerance mechanism against tissue autoantigens. Regulatory T cells (T regs ) are powerful suppressors of autoreactive T cells with high therapeutic potential (1-3). T regs also suppress auto-Ab production (4, 5). We recently showed in vivo that they do so in an antigen-specific (Ag-specific) manner (6, 7). These studies used rat insulin promoter HEL/OVA (ROH) mice expressing ovalbumin (OVA) and hen egg lysozyme (HEL) in pancreatic islet β-cells. Autoreactive OVA-and HEL-specific B cells, but not B cells specific for a foreign antigen, failed to proliferate in response to in vivo autoantigen (auto-Ag) challenge and instead underwent apoptosis in a strictly T reg -dependent fashion. T regs can affect B cells indirectly by suppressing the T-helper (Th) cells required for antibody production (8, 9). This did not rule out that T regs might also suppress B cells directly. Cell culture systems have revealed that CD25 + T regs can kill cocultured B cells (10-12). A recent in vivo study showed that T regs enter germinal centers and suppress B cells in this site (13,14). The question whether this occurred directly or indirectly remained open (15). This question is difficult to address in vivo because it requires an experimental system where T regs can suppress B cells but not Th cells.Another open question concerns the molecular mechanisms by which T regs suppress. In principle, T regs may suppress other T cells by (i) secreting inhibitory mediators; (ii) deprivation of survival factors; (iii) killing target cells by granzyme/perforin; and (iv) modulation of DCs by ligating inhibitory T-cell receptors (16,17). The exact contribution of these mechanisms in relevant in vivo situations and the mechanisms by which T regs suppress are unclear.Programmed death-1 (PD-1, CD279) is an activation-induced member of the extended CD28/CTLA-4 family that suppresses T cells (18-21). It has been associated with exhausted memory T cells in chronic viral infection (22,23) and with cytotoxic T-cell cross-tolerance (24). PD-1 has two known ligands, PDL-1 (B7-H1, CD274) and PDL-2 (B7-DC, CD273) (25,...

To study B-cell tolerance against non-lymphoid tissue autoantigens, we generated transgenic rat insulin promoter (RIP)-OVA/hen egg lysozyme (HEL) mice expressing the model antigens, OVA and HEL, in pancreatic islets. Their vaccination with OVA or HEL induced far less auto-Ab titers compared with non-transgenic controls. Depletion of CD25 + cells during immunization completely restored auto-Ab production, but did not affect antibodies against a foreign control antigen. Depletion at later time-points was not effective. OVA-specific CD25 + FoxP3 + T reg were more frequent in the autoantigen-draining pancreatic LN than in other secondary lymphatics of RIP-OVA/HEL mice. Consistently, B cells were suppressed in that LN and also in the spleen, which is known to concentrate circulating antigen, such as the antigens used for vaccination. Suppression involved preventing expansion of autoreactive B cells in response to autoantigen, reducing antibody production per B-cell and isotype changes. These findings demonstrate that CD25 + T reg suppress auto-Ab production against non-lymphoid tissue antigens in an antigenspecific manner.Key words: Auto-antibodies . B cells . Tolerance . Transgenic mice . T reg IntroductionAutoreactive B cells are controlled by a series of self-tolerance mechanisms. The first checkpoint incapacitates B cells of high affinity to autoantigen in the bone marrow, by receptor editing [1,2] or central deletion [1][2][3][4]. Self-reactive low-affinity B cells enter the periphery and can constitute up to 5-20% of circulating mature B cells in healthy individuals [5,6]. Therefore, further peripheral checkpoints exist, such as deletion [1,2,7] or anergy [1,8,9]. Anergic B cells are arrested in the transitional T2 developmental stage, followed by their death [10]. B cells also die in response to continuous antigen binding [11] and BCR signaling [9].Auto-Ab production by autoreactive B cells that survive these checkpoints can be avoided by extrinsic regulation [1], like the absence of T cell help resulting from central T-cell tolerance, which appears to be more efficient than central B-cell tolerance [12]. Th cells are required for B-cell survival [1], class switching, Ig synthesis and somatic hypermutation [6,13,14]. The latter process not only further increases BCR diversity but may also generate self-reactive B cells, with an even higher risk for autoimmunity, because after somatic hypermutation, B cells are long-lived and produce high-affinity Ab [15]. Thus, additional tolerance mechanisms seem to be required for maintaining peripheral B-cell tolerance, such as active suppression [6].In addition to T-cell suppression, T reg have also been proposed to control antibody production, since auto-Ab titers in B-cellmediated disease models were associated with decreased numbers or functionality of T reg , and because depletion of T reg aggravated disease [16,17]. The role of T reg in antibody production against non-lymphoid tissue autoantigens is unresolved. In rat insulin promoter (RIP)-mOVA mice expressing the mo...

Although the spleen is a major site where immune tolerance to circulating innocuous antigens occurs, the kidney also contributes. Circulating antigens smaller than albumin are constitutively filtered and concentrated in the kidney and reach the renal lymph node by lymphatic drainage, where resident dendritic cells (DCs) capture them and induce tolerance of specific cytotoxic T cells through unknown mechanisms. Here, we found that the coinhibitory cell surface receptor programmed death 1 (PD-1) on cytotoxic T cells mediates to their tolerance. Renal lymph node DCs of the CD8 + XCR1 + subset, which depend on the transcription factor Batf3, expressed the PD-1 cognate ligand PD-L1. Batf3-dependent DCs in the renal lymph node presented antigen that had been concentrated in the kidney and used PD-L1 to induce apoptosis of cytotoxic T cells. In contrast, T cell tolerance in the spleen was independent of PD-1, PD-L1, and Batf3. In summary, these results clarify how the kidney/renal lymph node system tolerizes the immune system against circulating innocuous antigens.

Regulatory T cells (Tregs) prevent autoimmunity but limit antitumor immunity. The canonical NF-κB signaling pathway both activates immunity and promotes thymic Treg development. Here, we report that mature Tregs continue to require NF-κB signaling through IκB-kinase β (IKKβ) after thymic egress. Mice lacking IKKβ in mature Tregs developed scurfy-like immunopathology due to death of peripheral FoxP3 Tregs. Also, pharmacological IKKβ inhibition reduced Treg numbers in the circulation by ∼50% and downregulated FoxP3 and CD25 expression and STAT5 phosphorylation. In contrast, activated cytotoxic T lymphocytes (CTLs) were resistant to IKKβ inhibition because other pathways, in particular nuclear factor of activated T cells (NFATc1) signaling, sustained their survival and expansion. In a melanoma mouse model, IKKβ inhibition after CTL cross-priming improved the antitumor response and delayed tumor growth. In conclusion, prolonged IKKβ inhibition decimates circulating Tregs and improves CTL responses when commenced after tumor vaccination, indicating that IKKβ represents a druggable checkpoint.

The NFκB transcription factor family facilitates the activation of dendritic cells (DCs) and CD4(+) T helper (Th) cells, which are important for protective adaptive immunity. Inappropriate activation of these immune cells may cause inflammatory disease, and NFκB inhibitors are promising anti-inflammatory drug candidates. Here, we investigated whether inhibiting the NFκB-inducing kinase IKK2 can attenuate crescentic GN, a severe DC- and Th cell-dependent kidney inflammatory disease. Prophylactic pharmacologic IKK2 inhibition reduced DC and Th cell activation and ameliorated nephrotoxic serum-induced GN in mice. However, therapeutic IKK2 inhibition during ongoing disease aggravated the nephritogenic immune response and disease symptoms. This effect resulted from the renal loss of regulatory T cells, which have been shown to protect against crescentic GN and which require IKK2. In conclusion, although IKK2 inhibition can suppress the induction of nephritogenic immune responses in vivo, it may aggravate such responses in clinically relevant situations, because it also impairs regulatory T cells and thereby, unleashes preexisting nephritogenic responses. Our findings argue against using IKK2 inhibitors in chronic GN and perhaps, other immune-mediated diseases.